1. Field of the Invention
The present invention relates, in general, to the fields of yeast transformation, yeast cells thereby transformed and production of recombinant products therefrom. More specifically, the present invention relates to the transformation of yeast by electroporation and spheroplast formation.
2. Background Art
Riboflavin (vitamin B2) is synthesized by all plants and many microorganisms, but is not produced by higher animals. Because it is a precursor to coenzymes such as flavin adenine dinucleotide and flavin mononucleotide, that are required in the enzymatic oxidation of carbohydrates, riboflavin is essential to basic metabolism. In higher animals, insufficient riboflavin can cause loss of hair, inflammation of the skin, vision deterioration, and growth failure.
The availability of means for the production of recombinant products, such as industrial enzymes, pharmaceutical proteins and products, vitamins, and cofactors, in eukaryotic systems such as yeast, provides significant advantages relative to the use of prokaryotic systems such as E. coli for the production of those products encoded by, or synthesized as a result of, recombinant nucleic acids. Yeast can generally be grown to higher cell densities than bacteria and are readily adaptable to continuous fermentation processing.
The development of yeast species as host/vector systems for the production of recombinant products is severely hampered by the lack of knowledge about transformation conditions and suitable means for stably introducing foreign nucleic acids into the yeast host cell. In addition, auxotrophic mutants are often not available, precluding a direct selection for transformants by auxotrophic complementation. New host/vector systems must be devised which facilitate the manipulation of nucleic acids as well as optimize the expression of inserted nucleic acid sequences so that the desired recombinant products can be prepared under controlled conditions and in high yield in yeast.
1. Methods of Transformation
a. Transformation by Electroporation
Becker et al. (Methods in Enzymology 194: 182–187 (1991)) relates to highly efficient methods of transformation of the yeast Saccharomyces cerevisiae. Becker also discloses spheroplast transformation.
Faber et al. (Curr. Genet. 25: 305–310(1994)) relates to a highly efficient method for transformation of the methylotropic yeast Hansenula polymorpha. Faber also applied the method to Pichia methanolica. 
Kasütske et al. (Yeast 8: 691–697 (1992)) relates to efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids.
Meilhoc et al. (Bio/Technology 8: 223–227 (1990)) relates to a high efficiency transformation system using intact Saccharomyces cerevisiae yeast cells and electric field pulses.
Piredda et al. (Yeast 10: 1601–1612 (1994)) relates to development of a transformation system for the yeast Yamadazyma (Pichia) ohmeri. 
Scorer et al. (Bio/Technology 12: 181–184 (1994)) relates to P. pastoris vectors allowing for rapid G418 selection of rare high copy number transformants for high level of expression in Pichia pastoris using both electroporation and spheroplast transformation systems.
Sherman et al. (Laboratory Course Manual for Methods in Yeast Genetics, pages 91–102, Cold Spring Harbor Laboratory (1986)) relates to a transformation of yeast mutants LEU2 and HIS3.
Thompson et al. (Yeast 14:565–571 (1998)) relates to an improved protocol for the preparation of yeast cells such as Saccharomyces cerevisiae and Candida albicans for transformation by electroporation.
Yang et al. (Applied and Environmental Microbiology 60(12): 4245–4254 (1994)) relates to high efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence ARS2. The method of transformation is by electroporation.
U.S. Pat. No. 5,716,808 to Raymond relates to methods for preparing Pichia methanolica cells containing foreign DNA constructs using electroporation and methods for producing foreign peptides in Pichia methanolica cells.
b. Transformation by Spheroplast Formation
Becker et al. (Methods in Enzymology 194: 182–187 (1991)) relates to highly efficient methods of transformation of the yeast Saccharomyces cerevisiae. Becker also discloses spheroplast transformation.
Scorer et al. (Bio/Technology 12: 181–184 (1994) relates to P. pastoris vectors allowing for rapid G418 selection of rare high copy number transformants for high level of expression in Pichia pastoris using both electroporation and spheroplast transformation systems.
U.S. Pat. No. 4,808,537 to Stroman et al. relates to a method for isolating and cloning methanol inducible gene from Pichia pastoris and the regulatory regions useful for the methanol regulation expression of heterologous genes. Stroman used spheroplast transformation.
U.S. Pat. No. 4,837,148 to Cregg et al. relates to autonomous replication sequences which are capable of maintaining plasmids as extra-chromosomal elements in host strains of Pichia. The patent further relates to constructs including the DNA sequences as well as transformed organisms produced by spheroplast formation. The patent additionally provides processes for producing the DNA sequences and constructs of the invention, as well as methods for isolating the sequences from any source.
U.S. Pat. No. 4,855,231 to Stroman et al. relates to DNA sequences which are responsive to the presence of methanol, catabolite non-repressing carbon sources and carbon source starvation. The '231 patent demonstrates spheroplast transformation of Pichia pastoris. 
U.S. Pat. No. 4,879,231 to Stroman et al. relates to a spheroplast transformation method for the yeast such as Pichia pastoris. 
U.S. Pat. No. 4,882,279 to Cregg et al. relates to a spheroplast transformation technique for yeasts of the genus Pichia. The '279 patent specifically embodies Pichia pastoris. 
U.S. Pat. No. 5,135,868 to Cregg relates to a method for the site specific genomic modification of yeasts of the genus Pichia. The '868 patent uses a spheroplast transformation method.
U.S. Pat. No. 5,268,273 to Buckholz relates to a method of spheroplast transformation of Pichia pastoris. 
U.S. Pat. No. 5,736,383 to Raymond relates to a method of transformation of yeast strains of the genus Pichia, particularly Pichia methanolica. The '383 patent further relates to a method of spheroplast transformation of yeasts of the genus Pichia as well as a method of transformation by electroporation.
c. Other Transformation Systems
Kunze et al. (Current Genetics 9(3): 205–209 (1985)) relates to a method of transformation of Saccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii G266. Saccharomyces cerevisiae, Candida maltosa and Pichia guilliermondii G266 have been transformed by the plasmid pYe(ARG4)411 which contains the S. cerevisiae ARG4 gene inserted into pBR322. Kunze used CaCl2 in the method of transformation.
Kunze et al. (J. Basic Microbiol. 25(2): 141–144 (1985)) relates to a method of transformation of the industrially important yeasts Candida maltosa and Pichia guilliermondii G266 using CaCl2.
Kunze et al. (Acta Biotechnol. 6(1): 28 (1986) relates to transformations of the industrially important yeasts Candida maltosa and Pichia guilliermondii. 
Neistat et al. (Mol. Ge. Mikrobiol. Virusol. 12: 19–23 (1986))(Abstract only) relates to transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeast by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae. The method of transformation is not disclosed.
U.S. Pat. No. 4,929,555 to Cregg et al. relates to a method for making whole cells of methylotrophic species of genus Pichia competent for transformation by DNA and a method for transforming with DNA whole cells of such species, particularly Pichia pastoris. 
U.S. Pat. No. 5,231,007 to Heefner et al. relates to a method of generating and isolating highly flavinogenic strains of Candida famata which produce riboflavin yields of around 7.0 to 7.5 grams per liter per 6 days. The method includes a combination of iterative mutagenizing steps and protoplast fusion steps performed on the parent strain and the descendent strains which are selected following each step according to a screening protocol.
2. Vectors, ARS Elements and Gene Libraries
Clyne, R. K. et al. (EMBO J. 14(24): 6348–6357 (1995)) relates to a fine structure analysis of ARS1, an ARS element of the fission yeast Schizosaccharomyces pombe. Characterization of a series of nested deletion mutations indicated that the minimal fragment of DNA encompassing ARS1 is large since no fragment under 650 bp retained significant ARS activity.
Liauta-Teglivets, O. et al. (Yeast 11(10): 945–952 (1995)) relates to the cloning of the structural gene of GTP-cyclohydrolase involved in riboflavin biosynthesis from a Pichia guilliermondii genomic library.
Cannon, R. D. et al. (Mol. Gen. Genet. 221(2): 210–218 (1990)) relates to isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae. 
Takagi, M. et al. (J. Bacteriol. 167(2): 551–555 (1986)) relates to construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.
Pla, J. et al. (Gene 165(1): 115–120(1995)) relates to ARS2 and ARS3 Candida albicans DNA fragments with autonomous replicating activity shown to promote non-integrative genetic transformation of both Candida albicans and Saccharomyces cerevisiae. 
U.S. Pat. No. 5,212,087 to Fournier et al. relates to ARS sequences which are efficacious in Yarrowia lipolytica as well as plasmids carrying these sequences.
U.S. Pat. No. 5,665,600 to Hagenson et al. relates to Pichia pastoris linear plasmids and DNA fragments thereof which contain ARS sequences. The '600 patent used the spheroplast transformation system as described in Cregg et al in U.S. Pat. No. 4,929,555.
U.S. Pat. No. 4,837,148 to Cregg et al. relates to autonomous replication sequences which are capable of maintaining plasmids as extrachromosomal elements in host strains of Pichia. The patent further relates to constructs including the DNA sequences as well as transformed organisms therewith. The patent additionally provides processes for producing the DNA sequences and constructs of the invention, as well as methods for isolating the sequences from any source.
3. Yeast Mutants
U.S. Pat. No. 5,120,655 to Foster et al. relates to strains of Candida famata which can produce 10 grams of riboflavin per liter in 6 days. The '655 patent also relates to strains having ATCC Accession Nos. 20849 and 20850. The '655 patent reports that riboflavin yields of more than 20 grams per liter in 200 hours have been achieved. The mutants were obtained by chemical or physical mutagenesis methods.
U.S. Pat. No. 5,164,303 to Heefner et al. relates to methods of producing riboflavin by culturing strains of yeast of the species Candida famata that produce at least about 10 grams of riboflavin per liter of fermentation medium in six days and culture methods therefore. The mutants were obtained by chemical or physical mutagenesis methods.
4. Riboflavin Production
U.S. Pat. No. 5,589,355 to Koizumi et al. relates to a process for producing riboflavin efficiently wherein riboflavin is formed and accumulated in a medium by culturing a microorganism carrying a recombinant DNA prepared from a microorganism belonging to the genus Corynebacterium or Brevibacterium. The transformed microorganism belongs to the genus Corynebacterium or Brevibacterium or Escherichia. 
Citation of documents herein shall not be construed as an admission that such documents are prior art to the present invention.
None of the above documents teaches methods for efficient transformation of flavinogenic yeast. There remains a need in the art for methods of transforming species of flavinogenic yeast and for using the transformed cells to produce economically important molecules such as enzymes, pharmaceutical and nonpharmaceutical proteins, vitamins, and cofactors. The inventors herein have recognized the problem in the art and developed transformation systems for flavinogenic yeast which have high efficiency of transformation and result in stable transformants.